1. The Field of the Invention
The present application relates to the use of automated imaging systems for high-content screening and analysis, collectively referred to as high-content imaging. More particularly, the present application relates to high-content imaging using a system having multiplexed imaging capabilities that include imaging of different chromophore labeled samples.
2. The Relevant Technology
A growing trend in microscopy over the last decade is the automated imaging of biological samples. Rather than the manual observation of samples, automated microscopy involves computer-controlled automatic selection and digital imaging of sample fields, enabling high throughput imaging of a large number of samples without end-user input.
Automated imaging is often known as HCI (High-Content Imaging) when applied to fluorescently labeled cells with automated quantitative analysis of the acquired images. In particular, HCI is a cell-based screening method that yields detailed information about the temporal-spatial dynamics of cell constituents and processes, and plays an important role in the use of cell-based screening for identification and validation of drug candidates. The information provided by HCI alleviates bottlenecks in the drug discovery process by providing deep biological information. The assays associated with this method use either fixed or live cells, depending on the biological information desired.
As an example HCI method of operation, the tissues or cells of interest are loaded onto a segment of a microscope slide or into an array of wells in a standard specimen plate (also known as a micro-titer or micro-well plate). The segment of the slide or specimen plate is then positioned on a stage within an imaging system so that the slide or specimen plate can move with the stage in both directions orthogonal to a configured light path of the system. Often the system includes a microscope for investigation of targeted cells. As a result, any of the individual wells or segment of the slide can be positioned in alignment with the microscope so as to be imaged through the microscope objective.
During a typical scan, the stage is moved by configured motors until one of the wells or segment of a slide is aligned with the objective and one or more of the cells within that well or segment of a slide are imaged through the objective. With respect to wells, the entire well can be imaged at the same time, or various fields within the well can be individually imaged.
When imaging is completed, the stage is then moved by the motors until another one of the wells or segment of a slide is aligned with the objective and, similar to the discussion above, one or more of the cells within the newly aligned micro-well or segment of a slide are imaged through the objective. This movement and imaging continues until all of, for example, the wells or defined segments of the slide have been imaged through the objective. Computerized analysis is then performed on the obtained images to determine information about the cells. This type of scanning can be performed many times a day for different HCI scans using the same machine.
HCI has mainly been applied on cells labeled with fluorescent probes, such as fluorescent ligands and immunofluorescent probes towards particular cellular targets, fluorescent environmental or cell state sensors, or fluorescent protein chimeras being endogenously expressed by the cell. For optimal signal-to-noise ratio detection, fluorescence typically requires using epifluorescence geometry of the imaging system, where the fluorescence signal retraces the same path as the illumination light, and the two are separated from each other by a wavelength discriminator such as a dichroic mirror, diffraction grating, or a solid-state discriminator such as an AOTF or LCTF. An added benefit to HCI is its multiplexed multispectral capability, where multiple fluorescent probes can be detected, each emitting fluorescence signal in a different color, as well as the ability to combine images acquired using white light brightfield imaging, which is an option available on most HCI platforms.
Brightfield microscopy uses a transmitted light geometry, wherein both the sample's illumination and the luminescent signals reaching the detector span the same wavelengths. However, the drawback to using brightfield microscopy when applied to biological samples is its poor contrast. Typically, the contrast can be enhanced by methods such as phase contrast and DIC (differential interference contrast) microscopy, where the sample illumination and the luminescent signal going to the detector still cover the same wavelengths.
An alternate approach to improve sample contrast and to distinguish specific cellular or tissue structures with transmitted light imaging is to stain the sample with different chromophores. Chromophores absorb light of specific wavelengths, and the differential absorption (i.e., subtractive mixing) and transmittance of transmitted light enhances the contrast of the sample. Additionally, depending on the different affinities various chromophores have for different areas of structures in cells and tissues, by the differential absorption of light by these chromophores these different cellular or tissue regions can be detected. The use of chromophores to differentially detect different cellular or tissue regions with high contrast, such as with H&E (hematoxylin and eosin) stains, is a routine and traditional use of microscopy in both life sciences research, as well as clinical diagnostic applications.
It is to be also noted that automated tissue scanners for digital pathology have the ability to image both fluorescence and chromophore absorbance but such methods are limited by either using multiple detectors to capture the primary colors or a color camera to capture the differential color absorptions by the chromophores. In addition, adding a color camera or multiple monochrome cameras can be expensive. Moreover, analysis of images acquired on a color camera, requires image processing algorithms that first unmix the different colors which is accomplished with the aid of reference images.
It is thus desirable to provide other cost-effective and automated systems and methodologies to improve sample contrast and to distinguish specific cellular or tissue structure that is complimentary to imaging of only fluorescently labeled cell types of automated imaging systems.
Accordingly, embodiments herein provide a system and method of High Content Imaging (HCI) on cell and/or tissue samples that are labeled with fluorescence probes and/or also labeled with chromophores. Since HCI is a multiplexed method where multiple different fluorescence colors, as well as white light brightfield imaging can be combined, the embodiments herein extend the capability of existing systems by including the ability to do multiplexed imaging with different chromophores using only a single detector.